Volume 391, Issue 1

Hexose-6-phosphate dehydrogenase (H6PD) is a luminal enzyme of the endoplasmic reticulum that is distinguished from cytosolic glucose-6-phosphate dehydrogenase by several features. H6PD converts glucose-6-phosphate and NADP + to 6-phosphogluconate and NADPH, thereby catalyzing the first two reactions of the pentose-phosphate pathway. Because the endoplasmic reticulum has a separate pyridine nucleotide pool, H6PD provides NADPH for luminal reductases. One of these enzymes, 11β-hydroxysteroid dehydrogenase type 1 responsible for prereceptorial activation of glucocorticoids, has been the focus of much attention as a probable factor in the pathomechanism of several human diseases including insulin resistance and the metabolic syndrome. This review summarizes recent advances related to the functions of H6PD.

Ecdysoneless (Ecd) is an evolutionarily conserved protein and its function is essential for embryonic development in Drosophila and cell growth in yeast. However, its function has remained unknown until recently. Studies in yeast suggested a potential role of Ecd in transcription; however, Ecd lacks a DNA-binding domain. Using a GAL4-luciferase reporter assay and a GAL4 DNA-binding domain fusion with Ecd or its mutants, we present evidence that human Ecd has a transactivation activity in its C-terminal region. Importantly, further analyses using point mutants showed that a single amino acid change at either Asp-484 or Leu-489 essentially completely abolishes the transactivation activity of Ecd. We further demonstrate that Ecd interacts with p300, a histone acetyltransferase, and coexpression of Ecd with p300 enhances the Ecd-mediated transactivation activity. Ecd localizes to both nucleus and cytoplasm and shuttles between the nucleus and cytoplasm; however, it exhibits strong nuclear export. Based on previous yeast studies and evidence provided here, we suggest that Ecd functions as a transcriptional regulator. Our results indicate an important function of human Ecd and provide a basis to explore the transcriptional partners of Ecd.

Binding of fluorine-containing drugs to bovine β-lactoglobulin, the most abundant whey protein in bovine milk, was investigated by means of 19 F NMR and mass spectrometry. The stoichiometry of the binding and its stability in acidic medium, where β-lactoglobulin is folded and stable, were also studied, along with competition from molecules that can be regarded as analogs of physiological ligands to bovine β-lactoglobulin. Conditional binding data were combined with protein structural information derived from circular dichroism and limited proteolysis studies. Spectroscopic techniques were also used to assess whether the bound drugs stabilize the protein structure against denaturation by chaotropes or temperature at various pH values. The results obtained provide evidence for the presence of multiple binding regions on the protein, with a specific and different affinity for structurally different classes of hydrophobic drugs and, more generally, that bovine β-lactoglobulin can bind and protect against low pH values various classes of drugs of pharmaceutical relevance.

Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P -3 is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.

Cytokinins are essential plant hormones that regulate numerous physiological processes. Recently, a protein was identified in mung bean ( Vigna radiata ) and characterized as a cytokinin-specific binding protein (VrCSBP). Fluorescence correlation spectroscopy was used to investigate the interaction between VrCSBP and its ligands. The synthetic cytokinin, N -phenyl- N ′-(4-pyridyl) urea, was labeled with two fluorophores, 7-nitro-2,1,3-benzoxadiazole and rhodamine B. Protein-ligand binding was analyzed in an equilibrium saturation binding experiment and confirmed by the competition assay. Surprisingly, it was found that VrCSBP binds not only to cytokinins, but also to gibberellins. In addition, in the presence of natural cytokinins and gibberellins, two populations of VrCSBP that differ in their diffusion coefficients were detected. The diffusion coefficients of these two populations could be related to mono- and dimeric states, which suggests a new mode of operation in ligand binding by VrCSBP, in which dimerization induced by natural ligands enhances the ligand binding capacity of the protein.

During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.

The mechanism of action of phytosphingosine (PHS), a member of the sphingosine family which has candidacidal activity when added externally, was investigated. Previously, it has been reported that the fungicidal activity of PHS is based on the induction of caspase-independent apoptosis. In contrast, we found that addition of PHS causes a direct permeabilization of the plasma membrane of yeast, highlighted by the influx of the membrane probe propidium iodide, and the efflux of small molecules (i.e., adenine nucleotides) as well as large cellular constituents such as proteins. Freeze-fracture electron microscopy revealed that PHS treatment causes severe damage of the plasma membrane of the cell, which seems to have lost its integrity completely. We also found that PHS reverts the azide-induced insensitivity to histatin 5 (Hst5) of Candida albicans . In a previous study, we had found that the decreased sensitivity to Hst5 of energy-depleted cells is due to rigidification of the plasma membrane, which could be reverted by the membrane fluidizer benzyl alcohol. In line with the increased membrane permeabilization and ultrastructural damage, this reversal of the azide-induced insensitivity by PHS also points to a direct interaction between PHS and the cytoplasmic membrane of C. albicans .

Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro . Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4α, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.

Transforming growth factor β1 (TGFβ1) has been implicated as a key contributor of immunosuppression in seminal plasma. The biochemical mechanisms that lead to production of active seminal TGFβ1 are not fully understood. It is plausible that TGFβ1 activation is partly induced simultaneously with the release of motile spermatozoa following liquefaction of the semen coagulum. Several members of the kallikrein-related peptidase (KLK) family are involved in the regulation of semen liquefaction. This study examines the involvement of these KLKs in TGFβ1 activation in vitro and ex vivo , in seminal plasma. Latent TGFβ1 was rapidly activated by KLK14. The latency-associated propeptide (LAP) was shown to be cleaved by KLK14 into small peptide fragments, providing a possible mechanism for TGFβ1 activation. KLK14 also cleaved the latent TGFβ binding protein 1 (LTBP1). KLK1, 2, and 5 might also contribute to TGFβ1 activation by nicking the LAP motif and inducing conformational changes that aid in subsequent processing of LAP or through LTBP1 cleavage. Our study provides strong evidence for the involvement of multiple members of the seminal KLK cascade in activation of latent TGFβ1 in seminal plasma. These findings might have clinical implications in infertility treatment of cases with concurrent delayed liquefaction and TGFβ1-related semen antigenicity.

Bacterial infections often upregulate the plasma kinin-forming cascade of the host (the ‘contact system’) which is triggered by adsorption of high molecular mass kininogen (HK), coagulation factor XII (FXII) and prekallikrein (pHPK) on the host or pathogen cell surfaces. A possible activation of the contact system upon infection of the human host by major fungal pathogens of Candida species has not been extensively explored until a recent report of tight binding of HK to the cell walls of these fungi. In the current study, the adsorption of the other contact system components to the cell surfaces of Candida albicans and Candida tropicalis was characterized. FXII was found to be tightly bound by Candida germ tube forms, to a level 5-fold higher than that for HK. In contrast, pHPK bound poorly but its additional amounts could dock to the cell wall through the surface-bound HK. It was also shown that within the complex of these proteins assembled on the cell walls of fungal hyphae, pHPK could be activated by FXIIa and the active HPK effectively produced kinins from HK. It is suggested that kinins, released at the Candida cell wall, can promote host colonization by the pathogen and the development of infection.

Proteases of Tannerella forsythia , a pathogen associated with periodontal disease, are implicated as virulence factors. Here, we characterized a matrix metalloprotease (MMP)-like enzyme of T. forsythia referred to as karilysin. Full-length (without a signal peptide) recombinant karilysin (49.9 kDa) processed itself into the mature 18-kDa enzyme through sequential autoproteolytic cleavage at both N- and C-terminal profragments. The first cleavage at the Asn14-Tyr15 peptide bond generated the fully active enzyme (47.9 kDa) and subsequent truncations at the C-terminus did not affect proteolytic activity. Mutation of Tyr15 to Ala generated a prokarilysin variant that processed itself into the final 18-kDa form with greatly reduced kinetics. Inactive prokarilysin with the mutated catalytic Glu residue (E136A) was processed by active karilysin at the same sites as the active enzymes. Karilysin proteolytic activity and autoprocessing were inhibited by 1,10-phenanthroline and EDTA. Calcium ions were found to be important for both the activity and thermal stability of karilysin. Using CLiPS technology, the specificity of karilysin was found to be similar to that of MMPs with preference for Leu/Tyr/Met at P1′ and Pro/Ala at P3. This specificity and the ability to degrade elastin, fibrinogen and fibronectin may contribute to the pathogenicity of periodontitis.

Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe -based whole-cell biotransformation now allows its use as a reference substance in doping control assays.